Smooth muscle (SM) cells form the main part of the walls of blood vessels, the airways, the gastrointestinal and reproductive tracts. Pathology of SM contractility plays a key role in hypertension, cerebral and coronary vasospasm, erectile dysfunction, bronchial asthma, and other diseases. SM contractility at a given level of Ca2+ is critically modulated by a complex network of protein-protein interactions, which can enhance the contractile effect acting via a small GTPase RhoA. The design of molecules that would alter these interactions could provide a more specific way of therapeutic targeting of RhoA signaling. It is well understood that RhoA, a ubiquitous molecular switch, is controlled by many different GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins), which either load RhoA with GTP (GEFs) or downregulate it by catalyzing the hydrolysis of GTP to GDP (GAPs). Which GEFs and which GAPs are active in SM, and how they contribute to the regulation of contractility - is not known. We propose to identify GEFs and GAPs active in SM, and to dissect the mechanisms by which they operate. This is an exciting stage in our ongoing studies of the mechanisms underlying the molecular and structural biology of the RhoA-dependent signaling pathways. Among the GEFs relevant to SM physiology are three RGS RhoGEFs, interacting with the Ga12/13 subunits, for which we have already accumulated a substantial amount of structural information. We present, for the first time, biochemical and functional data implicating p63RhoGEF/GEFT that interacts with Gaq11 linked to specific G-protein-coupled receptors. Promising results of our qRT-PCR experiments identify several GAPs and GEFs new to SM that may down and up regulate RhoA respectively and modulate SM contractility. We also formulate a new hypothesis, supported by preliminary data, which postulates that negative control is exerted on RhoA by cyclic nucleotides (cAMP) acting via the Rap1 GEF, Epac and Rap1 (another GTPase) to activate RhoA specific GAPs including ARAP3 and RA-RhoGAP. We will use a synergistic, multidisciplinary approach that bridges molecular physiology with structural biology. We will study SM tissues from normal and knock-out mice, with an experimental design that allows for the decoupling of the Ca2+-dependent phenomena from RhoA dependent regulation. Using X-ray crystallography, NMR, SAXs and DXMS, we will dissect the molecular mechanism by which the multidomain GEFs and GAPs are regulated in vitro and in vivo. Our research will explain fundamental aspects that control SM contractility and this knowledge may be used to design novel therapies for widespread diseases such as hypertension and asthma.